The recent severe acute respiratory syndrome (SARS) epidemic, which affected over 30 countries, resulted in more than 8000 cases of infection and more than 800 fatalities (World Health Organization, http://www.who.int/csr/sars/country/en/). A novel coronavirus was identified as the aetiological agent of SARS [1]. Analysis of the nucleotide sequence of this novel SARS coronavirus (SARS-CoV) showed that the viral genome is nearly 30 kb in length and contains 14 potential open reading frames (ORFs) [2–4]. These viral proteins can be broadly classified into 3 groups; (i) the replicase 1a/1b gene products which are important for viral replication, (ii) the structural proteins, spike (S), nucleocapsid (N), membrane (M) and envelope (E), which have homologues in all known coronaviruses, and are important for viral assembly, and (iii) the "accessory" proteins that are specifically encoded by SARS-CoV. Much progress have been made in characterizing these SARS-CoV proteins [5, 6], but the molecular determinant for the severe clinical manifestations of SARS-CoV infection in contrast to the mild diseases caused by most coronaviruses, remains to be determined. In addition, the exact roles of "accessory" proteins of SARS-CoV are still poorly understood.

The subject of this hypothesis relate to the S protein and one of the "accessory" proteins, the SARS-CoV 3a protein. The S protein, which forms morphologically characteristic projections on the virion surface, mediates binding to cellular receptor and the fusion of viral and host membranes, both of these processes being critical for virus entry into host cells [7, 8]. As such, S is known to be responsible for inducing host immune responses and virus neutralization by antibodies [9, 10]. 3a (also termed ORF3 in [2] and [11], as X1 in [3], and as U274 in [12, 13]) is the largest "accessory" protein of SARS-CoV, consisting of 274 amino acids and 3 putative transmembrane domains. Three groups independently reported the expression of 3a in SARS-CoV infected cells [13–15] and it was also detected in a SARS-CoV infected patient's lung specimen [14]. Antibodies against 3a were also found in convalescent patients [11, 12, 14].

This article hypotheses that the endocytotic properties of 3a allow it to modulate the surface expression of S and explores a functional significance for the interaction between S and 3a, which has been observed experimentally [13, 15].

The cellular fate of the S protein has been well mapped [16, 17]: S is cotranslationally glycosylated and oligomerized at the endoplasmic reticulum. Its N-linked high mannose side chains are trimmed, modified and become endoglycosidase H-resistant during the transportation to the Golgi apparatus. Only this fully-matured form of S can be assembled into virions and/or transported to the cell surface. The latter could cause cell-cell fusion and the formation of syncytia. Recently, Schwegmann-Wessels and co-worker reported that a novel sorting signal for intracellular localization is present in the S protein of most coronaviruses, but absent from SARS-CoV S [18]. Site-directed mutagenesis studies confirmed that a YxxΦ motif (where x is any amino acid and Φ is an amino acid with a bulky hydrophobic side chain) retains the S protein of TGEV intracellularly when it is expressed alone. On the other hand, SARS-CoV S is transported efficiently to the cell surface unless such a motif is introduced into its cytoplasmic tail by mutagenesis.

The YxxΦ motif has been implicated in directing protein localization to various intracellular compartments [19–21]. Furthermore, most YxxΦ motifs are capable of mediating rapid internalization from the plasma membrane into the endosomes. Interaction between the adaptor protein complex 2 (AP-2) with the YxxΦ motif present in the cytoplasmic domain of the internalizing protein concentrated the protein in clathrin-coated vesicle, which then budded from the plasma membrane resulting in internalization. However, it appears that the YxxΦ motif can also bind other adaptor protein complexes, like AP-1, 3 and 4, and the differential binding to the different adaptors will determine the pathway of a cargo protein containing a particular YxxΦ motif [21]. Coincidently, a YxxΦ motif in the cytoplasmic domain of 3a has previously been identified [13]. Furthermore, the juxtaposition of the YxxΦ motif and a ExD (diacidic) motif was found to be essential for the transport of 3a to the cell surface, consistent with the role of these motifs in the transportation of other proteins to the plasma membrane [22]. 3a on the cell surface can also undergo internalization [13].

Analyzing the experimental results present in these publications collectively, it is possible to postulate a functional role for the evolution of the SARS-CoV 3a protein. The SARS-CoV S protein lacks the YxxΦ motif but it can bind to the 3a protein which has internalization properties. In SARS-CoV infected cells, S is rapidly transported to the cell surface. But if 3a is expressed in the same cell, it is also transported to the cell surface where it can bind S. The interaction between 3a and S enables both proteins to become internalized, resulting in a decrease in the expression of S on the cell surface. Thus, this viral-viral interaction confers the functional role for the YxxΦ motif found in other coronaviruses to the SARS-CoV S. This hypothesis also implies that the precise mechanisms used by TGEV and SARS-CoV to reduce the expression of S are different although in both cases, the YxxΦ motifs will be crucial. In TGEV, the YxxΦ motif in S caused it to be retained intracellularly, while in SARS-CoV, S that is transported to the cell surface becomes internalized again after it interacts with the 3a protein.

Using mammalian cell culture system and biochemical methods, it will be possible to determine the exact effects of 3a on the trafficking properties of S. Mutagenesis studies can be used to map the protein domains that are important for the interaction between 3a and S and for the defining the manner by which 3a contributes to the reduction of cell surface expression of S. Given that a full-length infectious clone of SARS-CoV has been assembled [23], the use of reverse genetics would certainly reveal more about the interplay between 3a and S during SARS-CoV infection.

This hypothesis, if proven, will indicate that the interaction between SARS-CoV-unique 3a protein and S results in a reduction of S on the cell surface through the endocytotic properties of 3a [13]. During SARS-CoV infection, expression of S on the cell surface of an infected cell mediates fusion with un-infected neighboring cells, leading to syncytium formation. It follows that reducing the cell surface expression of S will delay this cell-damaging effect and prevent the premature release of unassembled viral RNA. It may also enhance virus packaging as it appears that the assembly of coronavirus occurs intracellularly, probably in the intermediate compartments between the endoplasmic reticulum and Golgi apparatus [24]. Clearly, this has certain advantages for the virus at certain stages of its life cycle. In addition, a reduction in the cell surface expression of S may also help the infected cell evade the host defense system and reduce the production of anti-S neutralizing antibodies. Conversely, host or viral factors that disrupt the interaction between S and 3a would favor the expression of S on the cell surface and enhance cell-cell fusion, a process that is important for viral spreading.

It therefore appears that the cell surface expression of S protein of SARS-CoV can be reduced like that for other coronaviruses, but the mechanism may be different. The trafficking of SARS-CoV S may be mediated through 2 separate viral proteins, expressed from separate subgenomic RNA, and regulated by numerous complex cellular processes including the efficiency of transcription and translation, post-translation modification and stability of the viral proteins, as well as their interactions with host factors. Indeed, it is crucial to determine how this unique pathway benefits replication of the SARS-CoV. It is also interesting to note that sequence comparison of isolates from different clusters of infection showed that both S and 3a showed a positive selection during virus evolution [29, 30], implying that these proteins play important roles in the virus life cycle and/or disease development and is consistent with the proposal that 3a has evolved to modulate the trafficking properties of the spike protein.

Yee-Joo Tan is responsible for the entire manuscript.